This invention relates to blood platelets and assays for determining platelet activation.
Upon activation, human platelets undergo changes in platelet surface membrane receptors, which may result in platelet aggregation, interaction with fibrin fibers, and the resulting formation of a thrombus. In certain disease states, e.g., coronary artery disease and diabetes, platelets may exist in a hyperreactive state, resulting in increased risk to the patient of thrombosis. Early detection of platelet hyperreactivity can permit the timely administration of antiplatelet drugs. Platelets may also exist in a hyporeactive state, placing a patient at risk of hemorrhage.
.alpha.-thrombin is among the most physiologically important activators of platelets. Quantitative determination of thrombin-induced changes in specific receptors, as measured by monoclonal antibody binding, has been carried out in assays performed on washed and resuspended platelets. Activation of platelets by adenosine diphosphate (ADP) and epinephrine has been measured in a whole blood assay by flow cytometry (Shattil et al. (1987) Blood 70:307-315).
It is known that the tetrapeptide glycyl-L-prolyl-L-arginyl-L-proline (GPRP), an analog of the amino terminus of the .alpha.-chain of fibrinogen and the fibrin monomer, binds to fibrinogen and, under some experimental conditions, inhibits fibrin polymerization.
U.S. Pat. No. 5,246,832 (issued September 21, 1993 to Michelson et al.) describes a method for measuring the thrombin reactivity of activated platelets in whole blood by flow cytometry. Total platelets are identified using an anti-platelet antibody labeled with a first fluorophore emitting light at a given wavelength. .alpha.-thrombin-activated platelets are then identified using a second, activated platelet-specific antibody labeled with a second fluorophore emitting light at a second, different wavelength. Thrombin-induced fibrin clot formation is inhibited by inclusion in the assay of GPRP (Michelson et al. (1991) Blood 77:770-779; Kestin et al. (1993) Circulation 88:1502-1511).
Platelet activation results in the shedding of small fragments of platelets, termed platelet-derived microparticles (PDMP). The surface of these PDMP provide the primary procoagulant surface, i.e., they are critical for the promotion of blood clotting. For example, PDMP bind coagulant factors V and VIII more avidly than platelets. PDMP appear to have a major role in the generation of procoagulant activity and, therefore, detection of PDMP is a useful indicator of procoagulant status (Sims et al. (1988) J. Biol. Chem. 263:18205-18212; Gilbert et al. (1991) J. Biol. Chem. 266:17261-17268). PDMP have been counted in a washed platelet system (Jy et al. (1992) J. Lab. Clin. Med. 119:334-345). Sims et al. (1988) supra and Gilbert et al. (1991) supra demonstrated that PDMP bind coagulant factors V and VIII, i.e., PDMP are procoagulant in a washed platelet system.
Prior art measurement of PDMP in whole blood (Abrams (1990) Blood 75:128-138) did not allow evaluation of the ability of platelets to generate PDMP in vitro in response to an agonist, because the addition of sufficient Ca.sup.++ for PDMP formation also results in stimulation of fibrin clot formation.